In situ hybridization (ISH) of tissue samples is a nucleic acid hybridization technique used to investigate and localize target nucleic acids in morphologically preserved structures, e.g. within a cell, a tissue, a nucleus or a chromosome.
Today most pathological samples are routinely fixed and paraffin-embedded to allow for histological analysis and for archival storage. Formalin fixation and paraffin embedding are estimated to be used in over 90% of specimens prepared by clinical labs in preparation of specimens for histological diagnosis. Archives of well-annotated formalin-fixed, paraffin-embedded (FFPE) tissue specimens are invaluable resources for retrospective studies of human diseases, however the FFPE procedure as well as the storage of the samples is known to introduce adverse effects on the RNA quality (Ahlfen et al. (2007) PLoS ONE 2(12): e1261). Formalin fixation proceeds relatively slowly, a fixation-time of 16-24 h is conventional, and results in a relatively slow quenching of the endogenous RNases which invariably cause some RNA degradation. However, numerous studies have shown the formalin fixation as well as other aldehyde-based fixations such as paraformaldehyde- and glutaraldehyde-fixed specimens can be used for in situ hybridization of RNA.
Several methods for ISH on formalin-fixed, paraffin-embedded tissue have been described; interestingly all protocols are comprised with a hybridization step in a formamide comprising hybridization buffer. Formamide is also mentioned as the component in the hybridization mix in EP 440.749 B1, EP 432.221 B1, U.S. Pat. No. 5,521,061 and U.S. Pat. No. 5,750,340 as well as in the papers on ISH prepared on archival FFPE specimens known to the inventor. However importantly, formamide is characterized as a teratogenic substance that should be avoided.
Recently, a large number of small non-coding RNA genes have been identified and designated as microRNAs (miRNAs or miRs) (for review, see Ke et al. 2003, Curr. Opin. Chem. Biol. 7:516-523). They are typically 17-24 nucleotides (nt) long RNAs that are processed from longer endogenous hairpin transcripts. To date more than 6000 miRs have been identified in humans, worms, fruit flies and plants according to the miR registry database release 11.0 in April 2008, hosted by Sanger Institute, UK.
The importance of microRNAs in cancer is highlighted in a recent article (Barbarotto et al 2008 Int. J. Cancer. 122:969-977), which summarizes the main paradigms for the miRNA involvement in human cancers: Thus, “(i) miRNAs are altered in every type of analyzed human cancer; (ii) miRNAs act as oncogenes and tumor suppressors; (iii) miRNAs alterations may cause cancer predisposition; (iv) miRNAs profiling is a new diagnostic tool for cancer patients and (v) miRNA profiling represents prognostic tools for cancer patients.”. Accordingly, methods in particularly ISH methods that can be used for localization of expression and quantification of microRNAs in specific cells and tissues from cancer patients are needed.
Some further of the recent attention paid to small RNAs in the size range of 17 to 25 nt is due to the phenomenon RNA interference (RNAi). RNAi is the mechanism in which double-stranded RNA leads to the degradation of any RNA that is homologous RNAi relies on a complex and ancient cellular mechanism that has probably evolved for protection against viral attack and mobile genetic elements. A crucial step in the RNAi mechanism is the generation of short interfering RNAs (siRNAs) which are double-stranded RNAs that are about 22 nt long each.
Quantification of microRNAs and siRNAs by ISH procedures is very challenging due to the small size of the RNAs. Furthermore a high specificity is required since different small RNAs may only differ with respect to a single nucleotide, but present day ISH-protocols for FFPE samples often suffer by lack of sensitivity or high background levels.
Thus an improved method of detecting small non-coding RNAs by ISH in archival paraffin embedded specimens is highly needed.
The present invention provides an improved, robust and fast ISH method for detection of non-coding RNAs in FFPE-samples. The method avoid use of the teratogenic formamide while providing even better ISH-results than obtained with standard formamide comprising hybridization buffers. Furthermore the method has the advantage that it can be used for quantification of small non-coding RNAs by ISH in archival FFPE-samples.